This invention relates to a method of analyzing multiple samples simultaneously by detecting absorption and systems for use in such a method.
The rapid development of biological and pharmaceutical technology has posed a challenge for high-throughput analytical methods. For example, current development of combinatorial chemistry has made it possible to synthesize hundreds or even thousands of compounds per day in one batch. Characterization and analysis of such huge numbers of compounds have become the bottleneck. Parallel processing (i.e., simultaneous multi-sample analysis) is a natural way to increase the throughput. However, due to limitations related to column size, pressure requirements, detector and stationary phase material, it will be very difficult to build a highly multiplexed high-performance liquid chromatography (HPLC) system. The same goes for building a highly multiplexed gas chromatography (GC) system.
High performance capillary electrophoresis (CE) has rapidly become an important analytical tool for the separation of a large variety of compounds, ranging from small inorganic ions to large biological molecules. With attractive features such as rapid analysis time, high separation efficiency, small sample size, and low solvent consumption, CE is being increasingly used as an alternative or complementary technique to HPLC. For example, the use of capillary gel electrophoresis has greatly improved DNA sequencing rates compared to conventional slab gel electrophoresis. Part of the improvement in speed, however, has been offset by the loss of the ability (inherent in slab gels) to accommodate multiple lanes in a single run. Highly multiplexed capillary electrophoresis, by making possible hundreds or even thousands of parallel sequencing runs, represents an attractive approach to overcoming the current throughput limitations of existing DNA sequencing instrumentation. Such a system has been disclosed in U.S. Pat. Nos. 5,582,705 (Yeung et al.), 5,695,626 (Yeung et al.), and 5,741,411 (Yeung et al.). In this system, light-induced fluorescence is exclusively employed as the detection method.
While fluorescence detection is suitable for DNA sequencing applications because of its high sensitivity and special labeling protocols, UV absorption detection has remained very useful because of its ease of imnplementation and wide applicability, especially for the deep-UV (200-220 nm) detection of organic and biologically important compounds. A capillary isoelectric focusing system using a two-dimensional CCD detector, in which one dimension represents the capillary length and the other dimension records the absorption spectrum, has been described by Wu and Pawliszyn, Analyst (Cambridge), 120, 1567-1571 (1995). The system has been used for two capillary tubes but is not easily adapted for three or more capillary tubes because the system requires the capillary tubes to be separated by space. Instead of providing wavelength resolution in the second CCD dimension, isoelectric focusing in two capillary tubes is simultaneously monitored. The use of optical fibers for illumination, however, has led to low light intensities and poor UV transmission. So, only visible wavelengths have been employed for the detection of certain proteins. Because the CCD has a very small electron well capacity (about 0.3 million electrons), the limit of detection (LOD) of this system is litnited by the high shot noise in absorption detection. The use of the CCD produces an overwhelming amount of data per exposure, limiting the data rate to one frame every 15 seconds. Also, the imaging scheme utilized is not suitable for densely packed capillary arrays because of the presence of mechanical slits to reict the light paths. Further, in order to avoid cross-talk, only square capillaries can be used.
Photodiode arrays (PDA) are used in many commercial CE and HPLC systems for providing absorption spectra of the analytes in real time. Transmitted light from a single point in the flow stream is dispersed by a grating and recorded across the linear array. A capillary zone electrophoresis system using a photodiode array as the imaging absorption detector has been described by Culbertson and Jorgenson, Anal. Chem., 7, 2629-2638 (1998). Different elements in the array are used to image different axial locations in one capillary tube to follow the progress of the separation. Because the PDA has a much larger electron well capacity (tens of million electrons), it is superior to the CCD for absorption detection. Time-correlated integration is applied to improve the signal-to-noise ratio (S/N).
What is still needed is an absorption detection approach for the simultaneous analysis of multiple systems. One such system is shown in U.S. Pat. No. 5,900,934 (Gilby et al.). This system includes a photodetector array comprising a plurality of photosensitive elements connected to provide a serial output. The elements are typically pixels of a photodiode array (PDA). The elements are illuminated by a light source positioned to illuminate at least a portion of the photodetector array. The light source may be an AC or DC mercury lamp or other useable light source for chromatography. An array of separation channels is disposed between the light source and the photodetector array, each of the separation channels having a lumen, a sample introduction end and a detection region disposed opposite the sample introduction end. The array is a multiple parallel capillary electrophoresis system. A mask element having at least one aperture for each associated separation channel is required. Each aperture corresponds to its associated separation channel, thereby selectively permitting light from the light source to pass through the lumen of its associated separation channel. At least a portion of the light passing through the lumen of the associated separation channel falls on a respective photosensitive element of the photodetector array to effectt masuremcnt of absorption of light by a sample introduced into the sample introduction end of the associated separation channel.
The system described by Gilby et al. has disadvantages because it limits the amount of light impinging on the separation channel, providing less than desirable light intensity to the PDA. Further, aligning the apertures and the mask elements with the separation channels, e.g., capillaries, is difficult for several reasons. For example, positioning the capillaries with equal separation there between is difficult as the capillaries generally are not of equal dimension, e.g., diameter tolerances vary greatly. Further, for example, the mask geometry does not provide identical light paths, which leads to nonlinear response. Also, a mask can produce stray light, which leads to poor detection limits, and does not completely eliminate crosstalk from the adjacent capillaries, since the light beams are diverging and cannot escape the detector element. In addition, a mask can be difficult to manufacture, due to the requirement of uniformity. Also, Gilby places the sample and the PDA too close together, resulting in stray light, cross talk and the inability to use the maximum pathlength of light.
Thus, in view of the disadvantages inherent to the methods and systems in the art, there remains a need a method of analyzing multiple samples simultaneously by absorption detection. It is an object of the present invention to provide such a method. It is another object of the present invention to provide a system for use in such a method. These and other objects and advantages of the present invention as well as additional inventive features will become apparent to one of ordinary skill in the art from the detailed description provided herein.
The present invention also addresses other disadvantages in the art. For example, since the invention of the polymerase chain reaction (PCR) in 1985 by Kary Mullis, the uiate in sensitivity, together with increasing ease in implementation, have placed this technique in a central position in molecular biology research and in clinical diagnosis (Rolf et al., PCR: Clinical Diagnostics and Research, 1992, Springer-Verlag, Berlin and Heidelberg). In the last ten years; PCR has stimulated numerous investigations in genetic analysis, and is even being used to determine the genetic basis of complex diseases (Sack et al., Science 230: 1350 (1985)). There is no need to reiterate the development of CE as a powerfuil analytical tool in post-PCR analysis. A large amount of research has been done to explore the advantages of CE over traditional slab gel electrophores, including high-speed, high-resolution restriction fragments analysis (Guttman et al., Anal. Chem. 62: 2348 (1992); Milofsky et al., Anal. Chem. 65: 153 (1993); Williams et al., J. Chromatogr. A680: 525 (1994); Chang et al., J. Chromatogr. B669: 113 (1995); Barron et al., Electrophoresis 16: 64 (1995); and Righetti et al., Anal. Biochem. 244: 195 (1997)), high-speed, high-throughput DNA sequencing (Ruiz-Martinez et al., Anal. Chem. 65: 2851 (1993); Lu et al., J. Chromatogr. A680: 497 (1994); Lu et al., J. Chromatogr. A680: 503 (1994); Fung et al., Anal. Chem. 67: 1913 (1995); Zhang et al., Anal. Chem. 67: 4589 (1995); Carrilho et al., Anal. Chem. 68: 3305 (1996); and Kim et al., J. Chromatogr. 781: 315 (1997)), rapid and precise DNA typing and sizing (Baba et al., Electrophoresis 16: 1437(1995); Noble, Anal. Chem. 67: 613A (1995); Zhang et al., Anal. Chem. 68: 2927 (1996); Isenberg et al., Electrophoresis 17: 1505 (1996); Zhang et al., J. Chromatgor. A768: 135 (1997); Butler et al., Electrophoresis 16: 974 (1995); and Wang et al., Anal. Chem. 67: 1197 (1995)), single-base mutation analysis (Marino et al., Electrophoresis 17: 1499 (1996); Arakawa et al., J. Chromatogr.: A664: 89 (1994); Hebenbrock et al., Electrophoresis 16: 1429 (1995); Kuypers et al., J. Chromatogr.: B 675: 205 (1996); Cheng et al., J. Cap. Elec. 2: 24(1995); and Ren et al., Anal. Biochem. 245: 9 (1997)) and the analysis of disease causing genes (Lu et al., Nature 368: 269 (1994); Felmlee et al., J. Cap. Elec. 2: 125 (1995); Gelfi et al., BioTechniques 19: 254 (1995); and Grossman et al., Nucleic Acids Res. 22: 4527 (1994)). In particular, capillary array electrophoresis, along with other micro-fabricated devices (Ueno et al., Anal. Chem. 66: 1424 (1994); Takahashi et al., Anal. Chem. 66: 1021 (1994); and Anazawa et al., Anal. Chem. 68: 2699(1996))are promising methods for the purpose of achieving high-throughput DNA analysis. In this regard, single capillaries have been utilized for DNA analysis (Guttman et al. (1992), supra).
The conventional protocol for DNA analysis calls for labeling with radionuclides or fluorescent tags before, during or after size-based separation in slab gel electrophoresis or in capillary gel electrophoresis (CGE). This derivatization process involves expensive reagents and raises safety concerns for the operator and for waste disposal because of the toxic nature of these labeling reagents.
The present invention can be applied to genetic typing and diagnosis based simply on UV absorption detection. The additive contribution of each base pair to the total absorption signal provides adequate detection sensitivity for analyzing most PCR products. Not only is the u ef specialized and potentially toxic fluorescent labels eliminated, but also the complexity and cost of the instrumentation are greatly reduced. The DNA analysis protocols can, therefore, be designed to take advantage of high-throughput capillary array gel electrophoresis and simple UV absorption detection, based on the inherent spectral properties of the DNA bases. UV absorption detection of DNA products reduces the cost of analysis, since it does not require labeling.
Similarly, peptide mapping represents one of the most powerful and successful tools available for the characterization of proteins (Garnick et al., Anal. Chem. 60: 2546-2557 (1988); Borman, Anal. Chem. 59: 969A-973A (1987)). Although less informative than protein sequencing, it allows rapid analysis with simple instrumentation. In peptide mapping, a sample protein is selectively cleaved by enzymes or by chemical digestion (Tarr et al., Anal. Biochem. 131: 99-107 (1983); Dong, Advances in Chromatography 32: 22-51, Marcel Dekker, Inc.: New York (1992); Geisow et al., Biochem. J. 161: 619-625 (1977); and Ward et al., J. Chromatogr. 519: 199-216 (1990)). The peptide map then serves as a unique fingerprint of the protein and can accurately reveal very subtle differences among individual variants. Trypsin is by far the most widely used proteolytic enzyme in peptide mapping. Its desirable feaares are that cleavage at the C-terminal side of lysine and arginine is generally quantitative under proper conditions and that trypsin tolerates concentrations of urea as high as 4 M (Dong (1992), supra). The disadvantage is that the fragments formed may be too small, averaging 7-12 amino acid residues, resulting in very complex tryptic maps. After tryptic digestion, the digest is typically analyzed by various methods, such as slab gel electrophoresis (Cleveland et al., J. Biol. Chem. 252: 1102-1106 (1977)), thin layer chromatography (TLC) (Stephens, Anal. Biochem. 84: 116-126 (1978)), HPLC (Hancock et al., Anal. Biochem. 89: 203-212 (1978); Cox et al., Anal. Biochem. 154: 345-352 (1986); Fullmer et al., J. Biol. Chem. 254: 7208-7212 (1979); Vensel et al., J. Chromatogr. 266: 491-500 (1983); Leadbeater et al., J. Chromatogr. 397: 435-443 (1987); Dong et al., J. Chromatogr. 499: 125-139 (1990); and Hartman et al. J. Chromatogr. 360: 385-395 (1986), and capillary zone electrophoresis (CZE) (Jorgenson et al., J. High Resolut. Chromatogr. Commun. 4: 230-231 (1981); Cobb et al., Anal. Chem. 61: 2226-2231 (1989); Chang et al., Anal. Chem. 65: 2947-2951 (1993); Nashabeh et al., J. Chromatogr. 536: 3142 (1991); Ward et al., J. Chromatogr. 519: 199-216 (1990); Janini et al., J. Chromatogr. 848: 417-433 (1999); Frenz et al., J. Chromatogr. 480: 379-391 (1989); and Grossman et al., Anal. Chem. 61: 1186-1194 (1989)) to yield a peptide map. Gradient reversed-phase HPLC is the most common form of peptide mapping in use today (Leadbeater et al. (1987), supra; Dong et al., (1990), supra; and Hartman et al. (1986), supra).
In particular, CZE has received considerable attention as a complementary method to reversed-phase liquid chromatography in peptide mapping efforts (Jorgenson et al. (1981), supra; Cobb et al. (1989), supra; Chang et al. (1993), supra; Nashabeh et al. (1991), supra; Ward et al. (1990), supra; Janini et al. (1999), supra; Frenz et al. (1989), supra; and Grossman et al. (1989), supra). Separation of various peptides can be optimized through pH adjustments. Through the addition of micelle-forming surfactants to the running buffer, a dynamic partition mechanism (i.e., hydrophobicity) of peptide separation can also be established for the neutral fragments. Although CE is quite efficient and fast for aawyzing peptide fragments, the complete separation of peptides in a digest of high molecular mass proteins, for example, is not possible by using a single buffer condition. Unlike HPLC, the implementation of gradient separation in CE is not trivial (Whang et al., Anal. Chem. 64: 502-506 (1992); and Chang et al., J. Chromatogr. B 608: 65-72 (1992)).
Although these methods are useful for characterizing proteins, there are still other problems, such as the relatively large amount of sample required, long analysis time, and efficiency of the derivatization reaction Also, a typical map contains 20-150 peaks, all of which should ideally be totally resolved (Dong et al. (1992), supra). Therefore, a high degree of column resolution and system precision are required to reproduce accurately the maps, preferably starting with subnanomolar quantities.
The present invention enables a peptide map to be obtained that can serve as a unique fingerprint of the protein. Reliable high-throughput analyses can be performed, for example, based on multi-dimensional CE and a single prescribed experimental protocol.
Combinatorial screening also has attracted much attention recently because of its ability efficiently and reliably to zero in and identify the best solution to a chemical or biochemical question (Borman, CandE News, Mar. 8, 1999, pages 33-60). In chemical synthesis, optimization of the reaction yield can be achieved by simultaneously exploring all possible reaction conditions, catalysts and reagents. In drug discovery, all related structural variants of a given candidate can be tested against the target. However, screening must be comprehensive so that there is no chance of missing the best combination. This dictates having a large number of experiments to cover many parameters and to extend the range of each of thee parameters. High throughput is a requirement in order to produce a timely result. It is primarily because of the advances in high-throughput technologies and automation that combinatorial screening became practical. Still needed are general and rugged analytical methodologies that can keep up with the large number of reactions that can be performed in any given time. Another issue is miniaturization of the entire operation. This impacts the cost of reagents, proper disposal of solvents, space for manipulation and storage, etc.
Currently, there are several parallel assays for screening homogeneous catalysts. Modifications in V absorption (Wagner et al., Sicence 270: 1797-1800 (1995); Menger et al., J. Org. Che. 63: 7578-7579(1998)), fluorescence (Cooper et al., J. Am. Chem. Soc. 120: 9971-9972 (1998); Shaughnessy et al., J. Am. Chem. Soc. 121: 2123-2132 (1999)), color (Lavastre et al., Chem. Int. Ed. 38: 3163-3165 (1999)) or temperature (Taylor et al., Science 280: 267-270 (1998); Reetz et al., Angew. Chem. Int. Ed. 37: 2647-2650 (1999)) induced by the catalytic reactions are indicators of catalytic activity. In these approaches, although the relative activity of the catalyst is determined quicldy, no quantitative information about the overall yield or the regioselectivity and stereoselectivity of the process can be obtained. It is also nesary that the product exhibit very different measurable properties compared to the solvent or the reagents. Most of the time, secondary screening is necessary. Mass spectrometry (MS) (Orschel et al., Angew. Chem. Int. Ed. 38: 2791-2794 (1999)), which also has been widely used to screen catalysts, can provide selective detection. However, to address stereoselectivity, these procedures still tend to be laborious (Reetz et al., Angew Chem. Int. Ed. 38: 1758-1761 (1999)). So far, MS is still a serial, rather than a pael, approach, although the analysis time is reasonably short.
Separation-based techniques can solve the above problems. Serial methods, which include HPLC and CE, have been used to analyze asynunetric catalysis (Porte et al., J. Am. Chem. Soc. 120: 9180-9187 (1998); Ding et al., Angew. Chem. Int. Ed. 38: 497-501 (1999)) and alkylation reactions (Gaus et al., Biotech. and Bioeng. 1998/1999 61: 169-177). The throughput that can be achieved with serial separation schemes is low even with special techniques, such as sequential sample injection (Roche et al., Anal. Chem. 69: 99-104 (1997)) and sample multiplexing (Woodbury et al., Anal. Chem. 67: 885-890 (1995)). Multiplex HPLC is another interesting approach (Gong et al., Anal. Chem. 71: 4989-4996 (1999)), but achieving a high degree of multiplexing, such as 96 capillaries in capillary array electrophoresis (CAE), is not trivial. Thin-layer chromatography and gel clectrophoresis, on the other hand, are difficult to completely automate.
A highly successfl format for combinatorial screening is that of DNA chips (Southern, Electrophoresis 16: 1539-1542 (1995); Chee et al., Science 274: 610-614 (1996); and Winzeler et al., Science 281: 1194-1197 (1998)). A comprehensive set of oligonucleotides immobilized within a snall area is used to identify specific target sequences by hybridization. Oligonucleotide chips also have been used to develop aptamers that exhibit specific protein-nucleotide binding (Weiss et al., J. Virol. 71: 8790-8797 (1997)). Such heterogeneous screening assays have benefited from sensitive detection based on laser-induced fluorescence (LIF), either by selective labeling or by selective quenching. For homogeneous assays, the 96-well microtiter plate is a popular format. Fluidic operations, plate readers and autosamplers to interface to stanard analytical instruments have been developed for this format. When there is a color (absorption) change or fluorescence change, detection and quantitation is straightforward. In many situations, however, the reaction mixture is complex and some degree of separation or purification is needed before measurement. Multiple liquid chromatographs or single instruments with several columns can in principle be used for analysis of the reaction mixtures. Still, much higher throughput and much smaller sample sizes, which means much smaller amounts of reagents, are desirable. The present invention enables such higher throughput and smaller sample sizes and does not require the species of interest to be fluorescent.
The present invention provides a method of analyzing multiple samples simultaneously by absorption detection. The method comprises:
(i) providing a planar array of multiple containers each of which contains a sample comprising at least one absorbing speies,
(ii) irradiating the planar array of multiple containers with a light source comprising or consisting essentially of at least one wavelength of light that is absorbed by one or more of the absorbing species, the absorption of which is to be measured, and
(iii) detecting absorption of light by one or more of the absorbing species with a detection means that is in line with the light source and is positioned in line with and parallel to the planar array of multiple containers at a distance of at least about 10 times a cross-sectional distance of a container in the planar array of multiple containers measured orthogonally to the plane of the planar array of multiple containers. The detection of absorbtion of light by a sample in the planar array of multiple containers indicates the presence of an absorbing species in the sample. The method can frther comprise:
(iv) measuring the amount of absorption of light detected in (iii) for an absorbing species in a sample. The measurement of the amount of absorption of light detected in (iii) indicates the amount of the absorbing species in the sample.
Also provided by the present invention is a system for use in the above method. The system comprises:
(i) a light source comprising or consisting essentially of at least one wavelength of light that is absorbed by one or more absorbing species, the absorption of which is to be detected,
(ii) a planar array of multiple containers, into each of which can be placed a sample comprising at least one absorbing species, and
(iii) a detection means that is in line with the light source and is positioned in line with and parallel to the planar array of multiple containers at a distance of at least about 10 times a crossectional distance of a container in the planar array of multiple containers measured orthogonally to the plane of the planar array of multiple containers.